Structural investigation of Trypanosoma cruzi Akt-like kinase as drug target against Chagas disease

According to the World Health Organization, Chagas disease (CD) is the most prevalent poverty-promoting neglected tropical disease. Alarmingly, climate change is accelerating the geographical spreading of CD causative parasite, Trypanosoma cruzi, which additionally increases infection rates. Still, CD treatment remains challenging due to a lack of safe and efficient drugs. In this work, we analyze the viability of T. cruzi Akt-like kinase (TcAkt) as drug target against CD including primary structural and functional information about a parasitic Akt protein. Nuclear Magnetic Resonance derived information in combination with Molecular Dynamics simulations offer detailed insights into structural properties of the pleckstrin homology (PH) domain of TcAkt and its binding to phosphatidylinositol phosphate ligands (PIP). Experimental data combined with Alpha Fold proposes a model for the mechanism of action of TcAkt involving a PIP-induced disruption of the intramolecular interface between the kinase and the PH domain resulting in an open conformation enabling TcAkt kinase activity. Further docking experiments reveal that TcAkt is recognized by human inhibitors PIT-1 and capivasertib, and TcAkt inhibition by UBMC-4 and UBMC-6 is achieved via binding to TcAkt kinase domain. Our in-depth structural analysis of TcAkt reveals potential sites for drug development against CD, located at activity essential regions.

The presented experiments offer primary insights into the structure and function of the central protein Aktlike of a protozoan parasite and furthermore reveal potential drug target regions crucial for TcAkt function but exhibiting significant structural differences to HsAkt.The detailed understanding of the mechanism of action of TcAkt forms the basis for the development of effective drugs against the expanding CD.

N-terminal TcAkt-PH forms a positively charged flexible cleft
The presented NMR solution structure of the membrane binding domain TcAkt represents to date the only experimentally determined structure of an Akt-like protein domain of a protozoan parasite.The structural determination of TcAkt-PH (11.7 kDa, 100 aa) was achieved by a combination of NMR experimental data including long range NOEs and CS-Rosetta 57 (Figs.1a, S1a, Tables S1, S2, and S3).The following structural analysis is based on the lowest energy structure of TcAkt-PH (PDB: 8OZZ) (Fig. 1b).
TcAkt-PH has a typical PH domain fold 46,59 consisting of a C-terminal α-helix and two antiparallel β-sheets, formed by four and three β-strands, respectively (Fig. 1b,c).All structural elements (strands β1-β7, helix α1) contribute to the formation of the hydrophobic core (Fig. S1) resulting in a stable structure with an averaged rotational correlation time of 8.68 ns (Eq.2), as observed by NMR relaxation experiments (Fig. S2a-d).Additionally, TcAkt-PH exposes an intense network of seven surface-exposed intramolecular salt bridges, providing further stabilization of the fold (Fig. S3).Expression constructs of C-terminal truncated TcAkt-PH (aa 1-95), results in a destabilization of the structure, probably due to missing helix-stabilizing residues S96 and K97.

Phosphoinositides bind into the basic cleft of TcAkt-PH
To analyze if TcAkt-PH binds to PIP ligands, we performed NMR CSP experiments using the soluble headgroup Ins(1,3,4,5)P 4 of the proposed ligand PI(3,4,5)P 3 (Figs.S4 and S5).The experiments confirmed an interaction of Ins(1,3,4,5)P 4 with TcAkt-PH exhibiting a dissociation constant of 40 ± 14 µM (Table S4).NMR 1 H- 15 N HSQC spectra of TcAkt-PH were recorded before and after the addition of increasing amounts of the ligand (Fig. S5).NMR chemical shifts are sensitive to their local chemical environment.Thereby, residues involved in direct interaction with a ligand can be detected via the degree of change of their chemical shift compared to the apo-form 52 .Residues directly located in the binding pocket generally exhibit a strong change of their chemical shift and/or peak intensity.Residues that do not bind to the ligand directly but experience ligand-induced conformational changes, can also be detected via CSP.For each amino acid of TcAkt-PH, the Euclidean distance or d-value was calculated (Eq.4) (Fig. 3a,b), defined as combined value of 15 N-and 1 H-shifts, thus representing the degree of change of the chemical shift of a specific residue upon ligand addition and its involvement in the ligand interaction (Fig. 3a,b).
Subsequent Molecular Dynamics (MD) simulations in combination with NMR experimental data reveal that the nature of the protein-ligand interactions between TcAkt-PH and Ins(1,3,4,5)P 4 is strictly polar including H-bonds, ionic interactions and water bridges (Fig. 3d, see SI Section 1, Fig. S7).Except for R38 and K19, all basic charged residues located in the positively charged cleft are interacting with the ligand (Figs.2a and S6).Additionally, N20 has profound interactions with Ins(1,3,4,5)P 4 , as well as Y34 and T47, which form water-mediated interactions (Fig. 3c,d).Interestingly, N20 faces away from the binding site in the apo-form of TcAkt but when bound to PIP 3 , N20 rotates towards the ligand, enabling interactions with PIP phosphate groups (Fig. 3b,c).

TcAkt PH domain undergoes local conformational changes while hosting Ins(1,3,4,5)P 4
For analyzing the impact of ligand interaction on TcAkt-PH dynamics, { 1 H}- 15 N heteronuclear NOE (hetNOE) experiments were recorded before and after Ins(1,3,4,5)P 4 addition (Fig. 4a).As shown in Fig. 4b, ligand binding induces a slight increase of rigidity of residues located at the binding site (loop regions β1-β2 and β6-β7) or close to the binding cleft of TcAkt-PH (strand β3 and loop β3-β4), whereas the dynamics of the rest of the protein are not affected (Fig. 4b,c).
As shown by MD simulations the increase of rigidity observed by NMR can be explained by ligand-induced loop-to-helix transitions.The apo-form of TcAkt-PH already reveals a tendency for loop-to-helix transitions (Figs.5a, S8a, and S9).However, binding of Ins(1,3,4,5)P 4 further stabilizes mentioned conformational changes for a more extended period (Figs.5a, S8b, and S9).
Although tested ligands bind into the same cavity of TcAkt-PH, ligand-induced conformational changes of TcAkt-PH loop regions are versatile and complex as shown in detail in Fig. 5a (Figs.S8 and S9).Phosphorylation patterns of PIP headgroups impact distinctive conformational changes of TcAkt loops β1-β2, β3-β4 and β4-β5, resulting in ligand-specific structural rearrangements of the PH domain, which subsequently could influence the versatile functionalities of TcAkt 43,44 .Still, all of the tested ligands induce a similar bending of loop β1-β2 when binding to TcAkt-PH, independent on their phosphorylation pattern (Fig. S11).

TcAkt-PH prefers PIP ligands with P3 and P5 phosphorylations
In humans, a binding preference of Akt1 PH domain to PI(3,4,5)P 3 and PI(3,4)P 2 26,32,42,64 was determined by monitoring its intrinsic tryptophane fluorescence upon binding to different inositol phosphates 65 , as well as by competitive HPLC binding experiments of 32 P-labeled phosphoinositides 66 .To analyze TcAkt binding preferences, we calculated the solvent-accessible surface area (SASA) and the buried surface area (BSA) of each of the tested ligands when bound to TcAkt-PH (Fig. S10d).The more buried a ligand is in the binding site, the larger its BSA will be.The extent of BSA increase is an important descriptor of ligand binding 67,68 and can therefore be used for analyzing the impact of different phosphorylation patterns of PIP ligands on the interaction with TcAkt-PH.
The calculated averaged BSA (Fig. S10d) shows no significant differences between all InsPs, reflecting that the depth of the binding pocket remains consistent and is equally accessible to all InsPs irrespective of the number and relative positions of phosphate groups on the inositol ring.However, when analyzing each phosphate position individually, different BSA values could be observed (Fig. S10c).Irrespective of the type of InsP, the P1 phosphate group has the lowest BSA, meaning that it is more exposed to the solvent relative to other phosphate positions.In the membrane-bound PIP ligand, the fatty acid tail is attached on phosphate group P1 and is therefore oriented towards the membrane rather than the binding site and does not seem to form specific interactions with TcAkt.
In comparison, the P3 phosphorylation has the highest BSA values in all InsPs.For Ins(1,4,5)P 3 , which lacks the P3 phosphate group, P5 seems to compensate for P3 with comparable BSA values.Thus, we propose a preference of TcAkt-PH for PIP ligands containing P3 and/or P5 phosphorylations.
As previously shown, MD data reveals that all PIP ligands induce a bending of loop β1-β2 (Fig. S11).Nevertheless, different phosphorylation patterns of PIP ligands initiate different local conformational changes of TcAkt and also vary in binding behavior, thus supporting a ligand specificity of TcAkt.In contrast to HsAkt1, which favors P3 and P4 phosphorylations of PIP 26,32,42,64 , TcAkt shows a preference for P3 and P5 phosphate groups.

The kinase domain of TcAkt contains conserved motifs essential for Akt activity
Since structure determination of full-length TcAkt by X-ray crystallography was not successful, we used AF 69 for calculating the structure of TcAkt (Figs. 6 and S12).According to internal quality parameters, the calculation runs were designated as successful (see Materials and Methods section ' AF calculations').
Crucial residues in regions associated with Akt activation are typically highly conserved among kinases and are also present in the AF TcAkt structure (Figs. 6 and S14): The glycine-rich G-loop in the kinase C-lobe is crucial for ATP binding and was stated to have the consensus sequence GXGXΦG, with Φ as hydrophobic residue 33,34,56 .While this is consistent with the G-loop in HsAkt1, in TcAkt the third glycine within this sequence is replaced by a serine (Fig. S14), an exception that is also found in other protein kinases 70,71 .The catalytic loop, also present in the C-lobe, is responsible for the phospho-transfer from ATP to the substrate and carries A value of 1 indicates that the respective interaction type persists throughout 100% of the simulation time, while a value greater than 1 signifies multiple instances of the same interaction type between corresponding residue and ligand over the simulation duration (e.g., multiple hydrogen bonds between arginine and the ligand).Additional details on the computed interactions (H-bonds, ionic interactions, and water bridges) can be found in the supplementary information (SI Section 1 'Detailed description for computed protein-ligand interactions of TcAkt-PH and Ins(1,3,4,5)P 4 shown in Fig. 3d').a conserved aspartic acid (in TcAkt D257) that interacts directly with the target S/T p-site (Hs: T308, Tc: T290) (Figs.S13 and S14) 27,34,56 .The activation loop starts with the highly conserved DFG motif and ends with the APE motif 33,34 , whereas the crucial threonine p-site (in T. cruzi T290) is located in between (Fig. S14).The h-motif is part of the C-tail and usually contains the sequence FXXF(S/T)(Y/F) 27 and a p-site.TcAkt shows a shortened version of the h-motif (FSF) including the putative p-site S450 (Figs.S13 and S14).Other p-sites in the C-tail that were linked to HsAkt1 activation (Hs: S477, T479) are absent in TcAkt 25,28,29 .

Full-length TcAkt retains kinase activity and possesses a similar ADP/ATP binding pocket compared to human Akt
To evaluate functional properties in a full-length context, we expressed and purified recombinant TcAkt (Fig. S15a) and tested its enzymatic activity in vitro using a polyclonal antibody that recognizes its phosphorylated form.The synthetic peptide RPRAATF was used as a substrate.As shown in Fig. S15b, TcAkt efficiently phosphorylates the threonine residue of the peptide substrate as did HsAkt3, which was used as a positive control.This indicates that TcAkt is capable of recognizing the consensus recognition motif RXRXX(S/T)f associated with Akt-mediated phosphorylation [35][36][37] .
Based on the presented AF structure, an ATP bound model of TcAkt was calculated using the recent release of RoseTTAFold All-Atom 72 .The protein-ligand model proposes that TcAkt binds ATP in a similar manner compared to HsAkt2 in complex with AMP-PnP, an ATP analog, and Mn 2+ (PDB: 1O6K) (Fig. 7), thus emphasizing a strong structural and sequential conservation in this region.Figure 7 shows the ATP binding site of TcAkt kinase domain with involvement of residues from the G-loop, the C-tail, the DFG motif, catalytic spine residues and shell residues 56,75,76 .According to Kornev et al. 76 catalytic spine residues of protein kinases, as well as the DFG motif, are essential for positioning ATP and Mn 2+ , while shell residues were identified to have a stabilizing effect 77 (Fig. 7).
Differential scanning fluorimetry (DSF) experiments revealed an increased melting temperature (Tm) upon ADP and ATP binding only in the presence of Mn 2+ (Figs.S16 and S17).These findings match observations reported by Pascuccelli et al. 71 , showing that TcAkt requires Mn 2+ to phosphorylate substrates in vitro, but is not dependent on Mg 2+ (Figs.S16 and S17).The Mn 2+ binding residues in the crystal structure of HsAkt2 (N293 and D280) align with the corresponding residues in TcAkt (N262 and D275), confirming a conserved binding mechanism of TcAkt and HsAkt (see SI Section 2).

Interdomain interface of TcAkt is established via hydrophobic and aromatic interactions
The interface between the kinase and the PH domain represents a favorable target for the inactivation of HsAkt1 due to its autoinhibitory functionality 25,26,[28][29][30] .To analyze TcAkt potential as drug target against CD, a detailed examination of this region is therefore of high interest.
The AF model of full-length TcAkt is present in a closed conformation forming interdomain interactions between the TcAkt-PH and the kinase domain (Figs.6 and 8c).The interface between loop β1-β2 of TcAkt-PH and the C-lobe of the TcAkt-kinase domain is constructed from a network of non-bonded interactions (33 contacts between 20 residues) (Figs.9d and S18) and has a surface area of 412-460 Å 2 .The tip of loop β1-β2 of TcAkt-PH carries two aromatic residues, F16 and Y17, which contribute to the hydrophobic aromatic cluster of the kinase domain built of F289, F291, F302 and Y340 (Figs. 8a, S14, and S19).F16 interacts directly with F291 and Y340, and additionally forms a weak π-stacking interaction with Y340 (ring-to-ring distance 4.2 Å)   (Fig. S19).The involvement of aromatic residues in the formation of the interdomain interface between loop β1-β2 of the PH domain and the C-lobe of the kinase domain is also observed in HsAkt1 (Figs. 9c and S19).

Model of PH domain-mediated Akt autoinhibition in T. cruzi
In HsAkt1 PIPs recruit the PH domain to the membrane by direct interactions, thus disrupting the autoinhibitory interface between the kinase and the PH domain 25,26,[28][29][30] .This conformational rearrangement exposes the activation loop and the h-motif of the kinase domain, thereby enabling phosphorylation of T308 and S473 and activation of Akt 26,30 .In the absence of PIPs, HsAkt1 stays in its inactive closed conformation, maintaining the autoinhibitory interface between the kinase and the PH domain 78 .
We propose a similar mechanism of action for TcAkt.As shown in the presented AF model, hydrophobic interdomain interactions between the activation loop of the kinase domain and loop β1-β2 of the PH domain, cause a closed conformation of TcAkt (Fig. 8a,c).In this conformation, the putative phosphorylation site of TcAkt T290 27 is not accessible, as it is shielded by the PH domain (Figs.8a and S13).Binding of the TcAkt-PH domain to a PIP headgroup induces a bending of loop β1-β2, as observed by MD simulations (Fig. S11).An opening of loop β1-β2 involves a change of the position of residue F16, which is thus pulled away from the interdomain interface as shown in Fig. 8b.As a consequence, the hydrophobic cluster of the kinase domain needs to be rearranged to shield the non-polar residues from the solvent exposure.This rearrangement of the region around the activation loop, including T290, could lead to an exposed position of T290, thereby enabling its phosphorylation and consequently the activity enhancement of TcAkt (Fig. 8b).The described disruption of the interface is accelerated as residues K76, T47, K15 and K36, previously involved in the interface, are switching interaction partners upon PIP contact (Fig. 8b,d).The disruption of the autoinhibitory interface consequently leads to an open conformation (Fig. 8d) that is associated with activation of Akt.

The interdomain interface of TcAkt differs from HsAkt
For evaluation of TcAkt's potential as drug target against CD, it is inevitable to characterize similarities and differences to its human ortholog in order to highlight putative regions for the inhibition of TcAkt.The following analysis concentrates on two activity essential regions: the autoinhibitory interface and the PIP binding site, both revealing clear structural and sequential differences between TcAkt and HsAkt1 as shown in Fig. 9. Overall, the presented AF model of TcAkt is in good alignment with the crystal structure of HsAkt1 (PDB: 7APJ) and the HsAkt1 AF model (AF-DB: AF-P31749-F1), where loop regions of the kinase domain are visible 25 (Fig. S12a,b).In contrast to the kinase domains which usually share a high conservation, the sequences of PH domains are generally more diverse among different species 45 .The sequence similarity (SS) of full-length sequences of TcAkt and HsAkt1 is 52.6%, while kinase domains share a 64.4% SS and PH domains have a SS of only 36.2% (Fig. S21).
As previously described, the interdomain interface of TcAkt represents a promising target for Akt inhibition.As shown in Fig. 9, the structural built of the interdomain interface differs significantly between HsAkt and TcAkt.Despite the distinctive structural arrangements, the interdomain interface of TcAkt and HsAkt1 is basically constituted via a hydrophobic interface, including an aromatic cluster (Figs.9c,d and S19).In both organisms, the hydrophobic tip of loop β1-β2 of the PH domain interacts with hydrophobic residues of the kinase domain (Fig. 9c,d).In TcAkt, the hydrophobic tip of β1-β2 in TcAkt is formed by residues F16 and Y17, compared to residues Y18 and I19 in HsAkt1.In HsAkt1, Y18 was stated to form a π-stacking interaction with F309 of the kinase domain 25,29 .In the presented AF model of TcAkt, a weak π-stacking interaction is established between F16 and Y340.In HsAkt1, functional relevant residues D323 and D325, as well as E17, have been described, which are involved in the interdomain interface 29 .Mentioned residues are not present in TcAkt.In HsAkt1 D323 and D325 form interfacial contacts to the PH domain (loop β6-β7, loop β3-β4, strands β1 and β2) and mutations result in HsAkt1 hyperphosphorylation 29 .HsAkt1 E17 forms a salt bridge with R86 stabilizing the autoinhibitory interface 29 .Oncogenic HsAkt1 mutant E17K results in enhanced membrane binding 29 .In TcAkt, the interdomain interface is not stabilized by salt bridges.TcAkt contains K15, at a similar position to HsAkt1 E17, but forming a hydrogen bond with F291.
The PIP 3 binding site of TcAkt is structurally different to HsAkt but is recognized by human PIP 3 competitor PIT-1 due to its basic charge A putative region for TcAkt inhibition represents the PIP binding site which regulates its activity and guides Akt function [42][43][44] .Despite two conserved basic residues (Tc: K11, R23; Hs: K14, R25), the binding clefts of HsAkt1-PH and TcAkt-PH are structurally and sequentially diverse as described in Fig. 9.The proposed consensus sequence KXn(K/R)XR located in loop β1-β2 and strand β2 to predict interactions with PIPs phosphorylated at position 3, matches with HsAkt1 but not with TcAkt 42,46 (Fig. S14).Despite the differences in the amino acid composition of the binding site, the binding mode to PIP ligands is also established in distinctive manners.In mammalian PH domains mainly strands β1 and β2 (including loop β1-β2) provide primary contact sites to PIP ligands 45,46 , involving also hydrophobic residues (loop β1-β2: Y18, I19) (PDB: 1UNQ) (Fig. 9a).In contrast, NMR and MD experiments performed with TcAkt-PH reveal strictly polar protein-ligand interactions and furthermore show that besides strands β1 and β2, also loops β3-β4 and β6-β7 are involved in PIP interaction (Figs. 3, 4, and 5).Interestingly, according to MSA analysis, the PIP binding site also differs among closely related Trypanosoma and Leishmania species (see SI Section 3, Figs. 10, S22, and S23) proposing that PIP binding mechanisms are species-specific.
PIT-1 is a non-phosphoinositide small molecule antagonist of PIP 3 that shows inhibition of Akt without affecting other PIP 2 -selective PH domains 32,54 .The PIT-1 binding site of human Akt overlaps with the PIP interaction area, involving residues W22, Y26, and N54 (N54 interacts with the phenyl group of PIT-1, W22 and Y26 interact with nitro-group of PIT-1) 54 .Using the presented AF model of TcAkt, we initiated docking experiments with human PIP 3 competitor PIT-1 revealing that the inhibitor binds to human and trypanosomal Akt in a similar manner (Fig. 11, see SI Section 4 'Docking studies of TcAkt and human Akt inhibitors capivasertib and PIT-1').The overall basic charge of the PIP 3 binding pocket seems to compensate for the sequential and structural differences of the PIP 3 interaction sites of human and trypanosomal Akt.

General Akt inhibitor capivasertib binds to TcAkt
One of the best-studied examples of ATP-competitive Akt inhibitors is capivasertib 55 .Capivasertib is a pyrrolo [2,3-d]pyrimidine derivative that acts as a pan-Akt inhibitor, inhibiting all human Akt isoforms, by binding into the ATP binding site.Only recently, capivasertib was approved by the FDA for breast cancer treatment, being the first ATP-competitive Akt inhibitor on the market 55,56 .Based on the calculated AF structure of TcAkt, we generated protein-ligand models of capivasertib to human and trypanosomal Akt using molecular docking approaches (Fig. 12, see SI Section 4 'Docking studies of TcAkt and human Akt inhibitors capivasertib and PIT-1').Although binding sites are similar between the proteins, TcAkt reveals a strong stereospecifity for (S)-capivasertib over (R)-capivasertib, which is not observed to that extent for HsAkt (see SI Section 4).Whereas in HsAkt E234, A230 and E228 are most profound residues for interaction with (S)-capivasertib and (R)-capivasertib, in TcAkt E218, L214 and D212 are responsible for the inhibitor's binding (Fig. 12).Further experiments may be needed for evaluating the inhibitory potential of capivasertib on TcAkt activity.

TcAkt inhibition by UBMC-4 and UBMC-6 involves the kinase domain
To date, two TcAkt inhibitors UBMC-4 23 and UBMC-6 24 have been defined, both detected via virtual screening and molecular docking approaches.Nevertheless, the lack of structural information restricts a detailed understanding of the inhibitory mechanisms.The presented AF-model enables the determination of the binding location of previously described TcAkt inhibitors, thus offering insights into their mechanism of action (Fig. 13).
UBMC-4 has been recently described as a potent TcAkt inhibitor in T. cruzi cell structures, targeting vital cellular processes and resulting in various severe effects, including apoptosis-like events 23 .It furthermore reveals relatively low cytotoxicity on human cell lines (LC50 > 40 μM) and effective absorption in mice models 23 .TcAkt inhibitor UBMC-4 was assumed to bind to the PH domain according to published MD studies 23 .Nevertheless, combining published data with the presented AF model, localizes the UBMC-4 binding region at the linker region between the PH and the kinase domain (R103, T108) and at the kinase domain (L131, D132, T203, K204, F435) (Fig. 13).The binding position of UBMC-4 suggests that inhibition of TcAkt is achieved by restricting the flexibility of the interdomain linker region und thereby preventing PIP induced conformational changes as well as an opening of TcAkt.

Discussion
The presented NMR structure, in combination with interaction experiments, MD simulations, Molecular Docking and AF-calculations, provides primary insights into the structure and function of a putative target against CD: TcAkt.The experiments reveal a PIP binding area, including ligand-induced versatile conformational changes of TcAkt regions, which likely guide the broad functionality of this central protein.The experimentally derived information combined with a full-length model of TcAkt and sequential analysis exposes activity essential regions of the proteins, leading to a proposed model of TcAkt activation via PIP-induced disruption of its interdomain interface.Detailed structural information reveil activity crucial regions of TcAkt with clear structural differences to the human ortholog HsAkt1 and thus highlights putative regions for targeting TcAkt inhibition.

Conformational changes induced by PIP ligands depend on phosphorylation patterns and play a crucial role in Akt activity
TcAkt binds PIP headgroups differing in phosphorylation of the inositol ring in the same binding pocket with comparable binding affinities but with clear preferences for P3 and P5 phosphorylations (Fig. S10).Phosporylation patterns of PIP headgroup inositol rings furthermore induce distinctive patterns of conformational changes, mainly regarding loop regions β1-β2, β3-β4 and β4-β5 of TcAkt-PH (Figs. 5a, S8, and S9).Nevertheless, all tested PIP headgroups result in an opening of loop β1-β2 upon binding to the PH domain, potentially inducing the disruption of the autoinhibitory interface (Fig. 8b).
Similarly, Ins(1,3,4,5)P 4 also stabilizes a loop-to-helix transition of residues 41-43 (SGP) in loop β3-β4 of TcAkt (Figs. 5a, S8, and S9).Other tested ligands did not induce these conformational changes.Three of the four tested ligands result in helical conformations of region 15-17 (KFY) located at loop β1-β2 at the direct binding site of PIP, whereas only Ins(1,3,5)P 3 destabilizes the helix transition.Destabilizing effects of Ins(1,3,5) P 3 lead to increased flexibility, which could enable interactions with other molecules or regulate functionality 84 .Nevertheless, further experiments are needed to determine the exact effect of local conformational changes on the activity of TcAkt.
The presented experiments show a complex pattern of diverse stabilizing or destabilizing effects of PIP ligands on TcAkt, proposing, similarly to HsAkt1, that the dynamics of loop regions of the PH domain are crucial for the functionality of TcAkt.Targeting the PIP-affected loop regions via binding of a ligand could offer another possibility for TcAkt inactivation by preventing subsequent structural changes essential for activity.

Potential strategies for TcAkt inhibition
HsAkt has undergone extensive investigation as a therapeutic target in oncology 85 and more recently it has been studied as drug target for treatment of cardiovascular diseases 86 , metabolic syndrome 54 , Parkinson's disease 87 and schistosomiasis, caused by the parasite Schistosoma mansoni 88 .To inhibit Akt kinases, several inhibitors have been developed based on various approaches, which potentially guide the way for TcAkt inhibition.ATP-competitive inhibitors target the active site in the open conformation of Akt kinases [89][90][91][92] .However, a high similarity of the ATP-binding site in TcAkt, HsAkt1 and potentially other AGC family kinases needs to be considered (Fig. 7) 93,94 .Presented docking studies could confirm that ATP-competitive inhibitor capivasertib recognizes human and trypanosomal Akt via similar binding areas (Fig. 12, SI Section 4).
Another target region for Akt inhibition represents the PIP-binding site of the PH domain.Small molecule antagonists of PIP 3 showed activity against PIP 3 -dependent PI3K/Akt signaling 54 supporting that this represents a promising region for Akt inhibition.Although the structure and sequence of the PIP binding region seems to be species-specific (Figs. 5, 9, 10, and 11), the general basic charge of this area may compensate for structural differences as shown by docking experiments with human PIP 3 competitor PIT-1 and human Akt and TcAkt (Fig. 11, SI Section 4).
Allosteric inhibitors bind to the inactive closed conformation of Akt by stabilizing the PH-kinase domain interface thus preventing Akt recruitment to the membrane and consequent Akt activation 89,95 .By targeting both domains, allosteric inhibitors reveal a high selectivity also among Akt isoforms [96][97][98] .Exploiting the presented significant differences between HsAkt1 and TcAkt interdomain interfaces (Fig. 9), this class holds potential for the development of TcAkt-specific inhibitors.

Conclusion
In 2019 the WHO established a 'World Chagas Disease Day' on 14 th of April in order to raise awareness for this neglected tropical disease and its global spread due to factors like climate change.The current lack of safe and efficient treatment in combination with the rising drug resistance of causative protozoan parasite T. cruzi, emphasizes the need for new strategies in order to fight this potentially chronic disease.This work offers a detailed analysis of a central protein of T. cruzi (Akt-like) as potential drug target against CD and includes atomic resolution data of activity essential regions, thereby forming the basis for structure-based rational drug design.
Cells containing full-length TcAkt protein were pelleted at 6000 xg, resuspended in 20 mL lysis buffer (50 mM Tris-HCl pH 8.0, 500 mM NaCl) supplemented with protease inhibitor cocktail containing 100 mM PMSF (BioBasic), 100 mM Benzamidine, 0.5 mg/ml Leupeptin, and 70 mg/ml Pepstatin A (BioShop), and sonicated on ice for 4 min (18% amplitude, 60 s on, 60 s off).The mixture was centrifuged at 26,800 × g at 4 °C for 1 h, and the soluble protein sample was subjected to Ni 2+ affinity chromatography (HisTrap, GE Healthcare Life Sciences).The column was washed with 5%, 7%, and 12% (v/v) imidazole of the elution buffer (50 mM Tris-HCl pH 8.0, 500 mM NaCl, 250 mM imidazole) 5 CV each.The protein was obtained with 100% elution buffer in 1.2 mL fractions.Then, the sample was diluted with 50 mM Tris-HCl pH 8.0 buffer to a final concentration of 125 mM NaCl, filtered using a 0.2 µm filter, and subjected to anion exchange chromatography on a Mono Q™ 10/100 GL column (Cytiva).The column was pre-equilibrated with buffer (50 mM Tris-HCl pH 8.0, 125 mM NaCl) and the protein was eluted with 500 mM NaCl using a stepwise gradient (125-500 mM) with a flow rate of 1.0 mL/min for 60 min.Pure protein fractions were collected, buffer exchanged (50 mM Tris-HCl pH 8.0, 125 mM NaCl), and concentrated using ultrafiltration devices (Amicon Ultra, MWCO 3000, Millipore).Finally, the concentrated protein fraction was loaded onto the SEC column, pre-equilibrated with buffer (50 mM Tris-HCl pH 8.0, 150 mM NaCl), and protein elution was performed utilizing the same buffer at a flow rate of 0.2 mL/min.
Protein concentration was determined by measuring the absorbance at 280 nm with the specific extinction coefficients for full-length TcAkt (ɛ = 55,700 M −1 cm −1 ) and TcAkt-PH (ɛ = 23,900 M −1 cm −1 ).The purity of the proteins was confirmed by SDS-PAGE, followed by Coomassie blue staining.

Akt activity assay
The kinase activity of purified recombinant TcAkt-6His was measured using a solid phase enzyme-linked immuno-absorbent assay kit (Abcam, ab139436), according to the manufacturer's instructions.Briefly, 200 ng purified protein was incubated with ATP (1 µg/µl) for 60 min at 30 °C.The phosphorylation of the synthetic peptide was detected with a phospho-specific substrate antibody incubated 60 min at 21 °C.Subsequently, multiple washes were performed, and anti-rabbit IgG:HRP conjugate (1 µg/mL) and TMB substrate were added.The colorimetric detection was measured at 450 nm in a spectrophotometer (Varioskan Flash Multimode Reader, Thermo Scientific).The human HsAkt3 was used as a positive control and kinase assay dilution buffer was used as blank.Each reaction was performed in triplicate, and the results were expressed as relative kinase activity.Data were analyzed using GraphPad Prism 8.0.1.

DSF experiments
The Tm of TcAkt-6His was determined by monitoring the fluorescence intensity of SYPRO Orange dye (Thermo Fisher) bound to protein as a function of temperature.The protein was diluted to 1.6 µM in a buffer containing 50 mM Tris-HCl (pH 8.0) and 150 mM NaCl in the presence or absence of the indicated divalent cations (2 mM MgCl 2 and 2 mM MnCl 2 • 4H 2 O) with Sypro 5 × at a final volume of 25 μl into the wells of a 96-well thin wall PCR plate.The nucleotides were evaluated at 16 μM.Thermal scanning (20-95 °C at 1.0 °C/min) was carried out in the real-time PCR equipment (CFX Connect, Biorad) measuring the intensity of fluorescence every 10 s with the SYBR channel.The melting temperature and the first derivative curve were calculated using the software of the equipment.Data are shown as mean from three independent experiments.Reactions without protein, in the presence of reaction buffer, and SYPRO, were included as controls.

Interproton distance restraints
Interproton distance restraints (NOEs) were obtained from a 3D 15 N-NOESY 99,100,117 (80 ms mixing time) and a 3D 13 C-NOESY (130 ms mixing time) experiment.NOE assignment was achieved by a combination of CYANAautomated NOE assignment 118,119 and manual assignment.Secondary structure predictions were done using backbone assignments and TALOS+ 58 (Fig. S24).
The plot reveals a clear funnel towards the lowest-energy model, thereby indicating that the CS-Rosetta structure calculation is converged.Ten structures were selected according to the lowest Cα-RMSD to the lowest-energy structure resulting in an averaged Cα-RMSD value of 1.7 Å, additionally confirming successful structure calculation.Refinement statistics were determined via the PSVS server (PMID: 17186527) (Table S3).The best-scored model (S_07667) was used for further structural analysis.

Calculation of electrostatic surface
The coulombic electrostatic potential was calculated from atomic partial charges and coordinates according to Coulomb's law: φ… potential, q…atomic partial charges, d…distances from the atoms, ε…dielectric constant.The resulting potential is in units of kcal/(mol•e) at 298 K. Standard amino acids are assigned atomic partial charges and types from the recommended force field versions in AmberTools 20 (for proteins: ff14SB) 61,62 .

NMR CSP experiments
For CSP experiments, a 170 µM 15 N-labeled sample and a 30 mM stock of Ins(1,3,4,5)P 4 in NMR buffer were prepared.Small volumes of Ins(1,3,4,5)P 4 were added stepwise to the protein sample up to a ratio of 1:35 (protein:ligand). 15N-HSQC spectra were recorded after each step to follow the shift changes.Spectra were analyzed using CcpNmr Analysis 2.4.2. 106.
A threshold value was determined according to the procedure described by Schumann et al. 120 to exclude residues with very small shift changes.
The 15 N chemical shifts were weighted with a scaling factor α = 0.14.The dissociation constant (K d ) was then fitted for each amino acid individually with the following equation: (1) ϕ = q i /εd i K d values with d-values below the calculated threshold (d-value < 0.008) were excluded from the dataset.Outliers were identified via boxplot analysis and excluded from the dataset (K d > 90 µM).Mean and standard deviation were calculated from the resulting 64 values (Table S4).

MD simulations
To model the TcAkt-PH structure with ligands (Ins(1,3,4,5)P 4 , Ins(1,4,5)P 3 , Ins(1,3,4)P 3 , Ins(1,3,5)P 3 ), we have used the experimental structure of the PH domain of HsAkt (PDB: 1UNQ) with bound Ins(1,3,4,5)P 4 as a template.The coordinates of the TcAkt-PH structure were aligned with the HsAkt-PH structure.Subsequently, using the builder tool in Maestro (Schrödinger, LLC) 121 , we have utilized the coordinates of the bound ligand to manipulate the structure of the original ligand and model other ligands with the aligned structure of TcAkt-PH.Subsequently, hydrogen atoms were added using the protein preparation wizard 122 in Maestro.Each modeled TcAkt-PH structure without ligands and with ligands then undergoes an energy minimization step only for the H atom, followed by solvation and neutralization.
For equilibration, the system was subjected to 100 ps of Brownian Dynamics NVT at 10 K with restraints on solute-heavy atoms, followed by short 12 ps NVT and 12 ps NPT at 10 K with restraints on solute-heavy atoms.Later, the temperature was increased to 300 K for another 12 ps NPT run with restraints on solute-heavy atoms.Finally, all restraints were removed, and a short 24 ps NPT run was performed, followed by another 1 µs long NPT at 300 K.
The equilibrated system was then further simulated for 1 µs long production runs at 300 K.In total, each system was simulated for 2 µs.For these simulations, the program Desmond 123 was used with the OPLS4 124 all-atom forcefield.A time step of 2 fs was used throughout the simulations, employing a Nose-Hoover 125,126 thermostat and a Martyna-Tobias-Klein 127 barostat, with relaxation times of 1.0 and 2.0 ps, respectively.The particle mesh Ewald 128 method was used to treat long-range interactions, and a nonbonded cutoff of 9.0 Å was used for short-range interactions.For analysis, the last 1 µs production run has been used.

AF calculations
Ab-initio models for TcAkt were calculated using an AlphaFold 2.3 installation in standard configuration for monomers with full databases and monomer model weights 69 .With the full-length sequence (aa 1-458) as input, 25 models were generated and ranked by the highest pTM score.The top-ranked model (see Fig. 6) reached a pTM score of 80.6.

RoseTTAFold All-Atom assembly calculation
The models of the TcAkt kinase domain bound to ATP were calculated on a RoseTTAFold All-Atom 72 installation in standard configuration.A set of 5 models was generated with the domain sequence (331 aa) and the chemical structure of ATP as input.All 5 predicted models show ATP at the same binding site.The models were ranked by the lowest pae_inter score, the top-ranked model reached a score of 7.6.The models were relaxed using amber99sb 129 and GAFF 130 force fields with parameters oriented to the relaxation algorithm of AlphaFold-Multimer 69,131 .The highest ranked model is shown in Fig. 7.

Molecular docking
The structure of HsAkt was retrieved from the RCSB PDB under accession code 4GV1 55 .Non-protein atoms were then removed from the structure, leaving only the bound inhibitor, Capivasertib.Subsequently, protein preparation was conducted using Maestro 121 which involved the addition of hydrogen atoms following an energy minimization process to refine the positions of the added hydrogen atoms.The coordinates of a modeled TcAkt structure were aligned with those of the 4GV1 structure, utilizing the bound inhibitor's coordinates for grid generation.A receptor grid was generated around the bound inhibitor to facilitate docking.The inhibitor molecule underwent a separate ligand preparation step to explore its possible conformations and stereoisomers.Generated ligand conformations were then subjected to docking into the receptor using the extra precision protocol of Glide software 132 .Furthermore, a similar procedure was repeated for docking the PIT-1 (CAS 53501-41-0) inhibitor with the PH-domain of HsAkt, employing both the PDB:1UNQ structure and the PH domain of the AF structure of TcAkt.

Figure 1 .
Figure 1.NMR derived structure of TcAkt-PH (PDB: 8OZZ) (a) CS-Rosetta 57,58 plot: All atom energies of TcAkt-PH models with respect to their Cα-RMSD values relative to the lowest-energy model.10 000 structures were calculated.For each structure the Cα-RMSD to the lowest-energy structure (S_07667) was calculated and plotted against the all-atom energy of each model.The run is designated as converged due to the shape of the plot and the averaged Cα-RMSD value of 1.7 Å of the final 10 structures to the lowest-energy model (S_07667).(b) Lowest energy structure as cartoon representation: TcAkt-PH forms a typical PH domain fold, built of two antiparallel β-sheets shown in blue (β1-β4, β5-β7) and a C-terminal α-helix α1 (red).Loops are shown in grey.(c) Topology and amino acid sequence of TcAkt-PH (aa 2-105).

Figure 3 .
Figure 3. TcAkt-PH PIP binding site evaluation by NMR and MD simulations.(a) Chemical shift perturbations of TcAkt-PH.The graph includes calculated d-values (see Eq. 4) for each amino acid of TcAkt-PH.(b) Surface display of TcAkt-PH colored by determined d-values according to a gradient.Residues exhibiting high d-values are shown in red (max: 0.69), low d-values in light yellow (min: 0.01), not affected residues below threshold (< 0.008) in white, n.a.residues (due to overlap or missing peaks) are shown in grey.(c) MD derived structure of TcAkt-PH interacting with Ins(1,3,4,5)P 4 (a frame was carefully chosen from the last 1 µs MD simulation to showcase the interacting residues adequately): Interacting residues are shown in sticks, contacts in dashed yellow lines, phosphate groups in red (ball representation).(d) H-bonds, ionic interactions, and water bridges plotted as interaction fractions for each interacting residue: Bar charts are normalized over the course of the trajectory.A value of 1 indicates that the respective interaction type persists throughout 100% of the simulation time, while a value greater than 1 signifies multiple instances of the same interaction type between corresponding residue and ligand over the simulation duration (e.g., multiple hydrogen bonds between arginine and the ligand).Additional details on the computed interactions (H-bonds, ionic interactions, and water bridges) can be found in the supplementary information (SI Section 1 'Detailed description for computed protein-ligand interactions of TcAkt-PH and Ins(1,3,4,5)P 4 shown in Fig.3d').

Figure 7 .
Figure 7. ATP bound to TcAkt and HsAkt2 kinase domains.TcAkt kinase domain (dark grey) bound to ATP (orange sticks) was calculated with RoseTTAFold 72 .HsAkt2 kinase domain (light grey) was complexed with ATP analog, AMP-PnP (not shown) (PDB: 1O6K) 73,74 (all-atom RMSD of aligned structures 0.883 Å).Zoomed details show ATP binding site.TcAkt residues are shown in dark sticks and HsAkt residues are shown in light sticks.Colors refer to specific regions of the kinase domain: Catalytic spine residues (turquoise), shell residues (red), DFG motif (blue), C-tail residues (yellow), G-loop (green), other residues interacting with ATP (grey).

Figure 8 .
Figure 8. Proposed model of TcAkt activation via disruption of its autoinhibitory interface upon PIPbinding.TcAkt PH domain is shown in turquoise, TcAkt kinase domain in sand and the C-tail in lavender.(a) Interdomain contacts between the hydrophobic tip of the PH domain (sticks colored in turquoise) and the kinase domain (sticks colored in sand) are shown as zoomed detail.Phosphorylation site T290 is colored in red.Conserved residues are marked with an asterisk (*).(b) Ligand induced bending of TcAkt loop β1-β2.Superposition of TcAkt apo-form (a) and PIP-bound TcAkt determined by MD simulations (pink).Red arrow indicates conformational changes upon binding to PIPs.(c) TcAkt is present in a closed conformation (inactive) and gets recruited to the membrane.The PH domain and the kinase domain are interacting via an autoinhibitory interface.(d) The binding of the TcAkt PH domain to PIP molecules in the membrane induces conformational changes of the hydrophobic tip (F16, Y17) of the PH domain loop β1-β2, leading to an opening of the structure and the disruption of the intramolecular autoinhibitory interface including a surface exposure of otherwise buried phosphorylation site T290.

Figure 9 .
Figure 9. Structural analysis of activity essential regions of TcAkt and HsAkt1.Due to high differences in the residual arrangements the structures are not superimposed.HsAkt PH domain is shown in green, HsAkt kinase domain in blue and the C-tail in grey.TcAkt PH domain is shown in turquoise, TcAkt kinase domain in sand and the C-tail in lavender.Flexible interdomain linkers are shown in light grey.PIP binding site: (a) PIP binding site of HsAkt1: PIP interacting residues are shown as green sticks (derived from the crystal structure of HsAkt1-PH in complex with Ins(1,3,4,5)P 4 ;PDB: 1UNQ) and presented on the apo structure (PDB: 1UNP) for comparative reasons.(b) PIP binding site of TcAkt: PIP interacting residues are shown as turquoise sticks (derived from NMR analysis and MD simulations) and presented on the apo structure (PDB: 8OZZ).Intramolecular interface: (c) Intramolecular interface of HsAkt1 as zoomed detail from full-length HsAkt1 (PDB: 7APJ): Involved residues of the kinase (HsAkt1-K: blue) and the PH domain (HsAkt1-PH: green) are shown as sticks.(d) Intramolecular interface of TcAkt as zoomed detail from full-length TcAkt (AF model): Involved residues of the kinase (HsAkt1-K: sand) and the PH domain (HsAkt1-PH: turquoise) are shown as sticks.Conserved residues are marked with an asterisk (*). https://doi.org/10.1038/s41598-024-59654-8

Figure 10 .
Figure 10.MSA derived conserved regions of Akt-like proteins in Trypanosoma and Leishmania species highlighted on full-length TcAkt (MSA: Figs.S22 and S23).Conserved residues are shown in red (in MSA residues are marked with an asterisk (*)), residues which have similar properties are colored in sand (in MSA residues marked with dots (:,.)), non-conserved residues are shown in grey.Residues involved in PIP interaction, are shown as sticks.(a) Conserved regions of Akt-like proteins among Trypanosoma spp.(Fig. S22) (b) Conserved residues in PIP binding site amongTrypanosoma spp.(c) Conserved regions of Akt-like proteins among Trypanosoma and Leishmania spp.(Fig. S23) (d) Conserved residues in PIP binding sites of Trypanosoma spp.and Leishmania spp.Further details are described in SI Section 3.

Figure 13 .
Figure 13.TcAkt inhibitors UBMC-4 and UBMC-6 binding sites mapped on the AF model of TcAkt.TcAkt PH domain is shown in turquoise, the flexible linker in grey, the TcAkt kinase domain in sand and the C-tail (including the h-motif) in lavender.P-sites are colored in red and shown as sticks.Ligand interacting residues of TcAkt are shown in green and presented as sticks.(a) UBMC-4 binding site determined by Bustamante et al. 23 (b) UBMC-6 binding site determined by Ochoa et al. 24 .